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Abstract

Background

Dopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson’s
disease (PD) in part because their oxidative environment in the substantia nigra (SN)
may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble
Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons
in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate
TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution
of ceramide sphingolipid signaling in TNF-dependent toxicity.

Results

Ceramide dose-dependently reduced the viability of DA neuroblastoma cells and primary
DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three
different inhibitors during TNF treatment afforded significant neuroprotection by
attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane
potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics
mass spectrometry we confirmed that TNF treatment not only promotes generation of
ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases
(DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range
reduced cell viability and inhibited neurite outgrowth and branching in primary DA
neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its
neurotoxicity in DA neurons.

Conclusions

We conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid
species that promote degeneration and caspase-dependent cell death of DA neurons.
Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective
strategies that can delay or attenuate the progressive loss of nigral DA neurons in
patients with PD.

Keywords:

Background

The exact molecular mechanisms that contribute to pathogenesis in Parkinson’s disease
(PD) have not been well delineated; many different cellular processes have been implicated
in PD, including diminished function of the ubiquitin proteasome system, generation
of reactive oxygen species, endoplasmic reticulum (ER) stress, compromised mitochondrial
function and protein aggregation (Reviewed by [1]). Additionally, inflammation and activated microglia have been generally implicated
in PD pathology [2-7] and increased levels of pro-inflammatory cytokines such as TNF, IL-1β and IL-6, have
been observed in the cerebral spinal fluid (CSF) and striatum of PD patients relative
to healthy age-matched controls [8]. Furthermore, gene polymorphisms in inflammatory genes (TNF-308 and IL-1β-511) have
been associated with an increased risk of developing PD [9]. Specifically, we have previously reported that blocking soluble TNF (solTNF) signaling
with novel dominant-negative TNF inhibitors attenuates loss of dopaminergic neurons
both in vitro and in vivo[10]. Soluble TNF signals through the canonical transmembrane death receptor TNF receptor
1 (TNFR1) to potently transduce inflammatory stimuli [11,12]. TNFR1 is constitutively expressed by most cell types, including DA neurons, which
are acutely sensitive to TNF-induced toxicity [13-15]. However, TNFR1 can elicit signaling through numerous down-stream effectors, including
p38, JNK, MAPK, and ceramide (Reviewed by [16]) but identification of specific pathways required for TNF-induced cytotoxicity in
DA neurons has not yet been forthcoming.

The aim of this study was to test the hypothesis that ceramide signaling cascades
are an important effector arm of TNF-mediated cytotoxicity in DA neurons. Ceramide
is a sphingolipid with a well-established role in cell membrane homeostasis [17]. However, a wealth of studies over the past decade established the role of ceramide
and its downstream metabolites as second messenger sphingolipids due to their rapid
and transient generation in cells and their ability to modulate a variety of physiologic
and stress responses [18-20]. Specifically, ceramide has been implicated in the cell death pathway activated by
the death domain receptor ligands TNF and Fas-L [21,22]. Additionally, ceramide has been shown to activate apoptosis in primary cortical
neurons [23] and in primary neuronal cultures from embryonic mesencephalon [24], but its role as a critical downstream effector of TNF-induced apoptosis in DA neurons
has not been fully delineated. To explore the role of ceramide signaling in the TNF-dependent
cytotoxicity of DA neurons, we used both primary neuronal cultures from embryonic
rat ventral mesencephalon and the MN9D dopamine neuron-like cell line [25] which is a hybridoma line derived from fusion of murine embryonic ventral mesencephalon
and neuroblastoma cells and is often used as an in vitro model of DA neurons [26,27] because the cells express high levels of tyrosine hydroxylase (TH), the rate limiting
enzyme in dopamine biosynthesis, and efficiently synthesize, store and release dopamine
[28]; additionally, their sensitivity to oxidative stress and inflammatory stimuli is
similar to that of primary DA neurons from ventral midbrain [25-27]. Here we report that TNF and ceramide exert dose-dependent cytotoxic effects on DA
neuroblastoma cells and primary DA neurons. Functionally, inhibitors of SMase activity
which block sphingomyelin hydrolysis and ceramide generation attenuated TNF-induced
cytotoxicity, decreases in phospho-Akt, increases in caspase 3 cleavage as well as
mitochondrial membrane potential changes, and ER stress in DA cells. Ultimately, the
mechanisms of TNF-induced cytotoxicity in DA cells culminated in and were found to
be completely dependent on caspase signaling, suggesting a model in which ceramide/sphingolipid
signaling cascades are key effectors of TNF-dependent apoptotic death in DA neurons.
Our data also revealed that TNF treatment not only activates sphingomyelinases (SMases)
to produce ceramide but also leads to generation of several other atypical deoxy-sphingoid
bases (DSBs) including desoxymethylsphingosine (1-desoxyMeSo), deoxysphinganine (deoxySa),
and desoxymethylsphinganine (1-desoxyMeSa); when added exogenously in vitro, some of these DSBs inhibit neurite outgrowth and are toxic to DA neurons. These findings suggest that multiple sphingolipid mediators may be responsible for
mediating TNF neurotoxicity and identification of specific sphingolipid metabolites
may reveal opportunities for drug development to delay or prevent DA neuron degeneration.

Experimental procedures

Primary and Cell Line Cultures

The MN9D dopaminergic neuroblastoma cell line was developed by Dr. Alfred Heller [25] and was a generous gift from Dr. Michael Zigmond, at the University of Pittsburgh.
MN9D cells were grown in culture in sterile complete media (CM) which consisted of:
high glucose (4,500 mg/L) Dulbecco’s Modified Eagle Medium (DMEM, Sigma, D5648) dissolved
in sterile tissue culture tested water (Sigma) supplemented with 10% fetal bovine
serum (FBS, Hyclone Fetal Clone III), sodium bicarbonate (3.7 g/L, Sigma), 25 mM HEPES
(Sigma), and 1% Penicillin/Streptomycin (Sigma) at a final pH of 7.3 in a humidified
5% CO2 incubator at 37°C. MN9D cell cultures were seeded in 75 cm2 tissue culture flasks (Costar) and plated at a density of 7,500 cells per well for
96-well plates (100 μL CM per well); 35,000 cells per well (500 μL CM per well) for
24-well plates; and 50,000 cells per well (2 mL CM per well) for 6-well plates. After
plating and allowing attachment of cells overnight in CM, MN9D cells were differentiated
for 72 hrs via a complete media change to differentiation media (DM) which contained
serum free DMEM (same CM as above, except FBS was excluded) supplemented with 5 mM
2-Propylpentanoic acid (valproic acid, Sigma, P6273) and 1X N2 supplement (Invitrogen)
as described in previous protocols [26].

MTS Metabolic Assays

Treated diff-MN9D cells in 96-well plates were evaluated for overall viability using
the MTS assay (Promega, CellTiter 96 AQueous One Solution Cell Proliferation Assay)
according to the manufacturer’s instructions. Twenty microliters (20 μL) of the MTS
reagent was added to cell cultures with DM-containing treatments and/or inhibitors.
The cells were incubated with the MTS reagent at 37°C, 5% CO2 for 2 hrs prior to colorimetric quantification of MTS reduction into a blue formazan
by-product by metabolically active cells. The absorbance of blue formazan was measured
at 492 nm wavelength using a Multiskan Ascent absorbance plate reader (Thermo Labsystems).

LDH Release Cytotoxicity Assay

Treated diff-MN9D cells in 96-well plates were evaluated for cytotoxicity using an
LDH release assay (Clontech Laboratories, Mountain View, CA) as per the manufacturer’s
instructions. LDH reactions were measured at a wavelength of 492 nm on an absorbance
plate reader (Thermo Lab Systems Multiskan Ascent). The maximum LDH activity was determined
by lysing the cells with 1% Triton X-100.

Neurite Length and Branching Studies

After treatment with the specified sphingolipids, primary rat mesencephalic (MES)
cultures were fixed with 4% paraformaldehyde, stained with anti-MAP2 (1:1000, Millipore,
MAB3418) and anti-TH antibodies (1:1000, Millipore, AB152) and counterstained by DAPI.
Images were captured on an IMAGEXPRESS 5000A automated cellular imaging and analysis
system. When analyzing the morphology of TH-positive neurons, the neurite outgrowth
application module of MetaXpress software was used and multi-parameter analysis measurements
were performed.

Sphingomyelinase and Ceramide Synthesis Inhibition

Diff-MN9D cells in 96-well plates were pre-treated in triplicate or quadruplicate
with one of four different pharmacological inhibitors: the acid sphingomyelinase (ASMase)
inhibitor desipramine HCl, used at 1 μM and 5 μM, (Sigma, D3900, dissolved in sterile
H2O), the neutral sphingomyelinase (NSMase) inhibitor GW4869, used at 10 μM, and 20 μM,
(Calbiochem, No. 567715, dissolved in DMSO, aliquotted and stored under argon gas),
the synthetic bisphosphonate sSMase inhibitor 7c, also known as ARC39, used at 1 μM,
(a generous gift from Dr. Christoph Arenz, Humboldt University in Berlin, Germany),
the serine palmitoyltransferase inhibitor myriocin (a generous gift from Dr. Philip
Scherer, UT Southwestern Medical Center at Dallas), dissolved in ethanol and used
at 10 μM or the ceramide synthase inhibitor Fumonisin B1, used at 50 μM (Axorra LLC,
No.: 350-017-M001, dissolved in sterile H2O). Diff-MN9D cells were pre-treated with ceramide inhibitors or control diluents
for 30 minutes via a 50% media change with DM that contained a 2X concentration of
the respective inhibitor or control diluent (i.e. 50 μL of the initial 100 μL DM was
removed and replaced with 50 μL of DM containing a 2X concentration of a ceramide
inhibitor for a final volume of 100 μL with 1X inhibitor). After pre-treatment with
ceramide inhibitors for 30 minutes, TNF was added by a 1:100 dilution of a TNF stock
concentration into media that contained ceramide inhibitors (1 μL of 100X TNF was
added to 100 μl DM). In the case of GW4869, which declines in effective NSMase-inhibition
over time [29] the GW4869 reagent was added to DM 30 minutes prior to TNF treatment by a 50% media
change and was then re-added 24 hrs after initial GW4869 pre-treatment by addition
of a 1:100 dilution of a GW4869 stock concentration (1 μL of 100X GW + 100 μL DM to
equal 1X) into DM already containing TNF treatments. Diff-MN9D cells were incubated
at 37°C, 5% CO2 for 48 hrs post TNF treatment prior to determination of cell viability by MTS assay.

Western blots for ER stress, caspase-3, and p-AKT activation

MN9D cells were plated on 6-well plates at the density of 50,000 cells/well. Twenty-four
to forty-eight hours later, the complete media was changed into differentiation media
and the cells were neurally differentiated for 72 hours. Before TNF treatment, diff-MN9D
cells were pre-incubated with desipramine or GW4869 for 1 hour. After 24- hours treatment
with ceramide, TNF, TNF/Des or GW, cell lysates were collected in 200 uL SDS-PAGE
loading buffer. When running SDS-PAGE, 15ul of sample lysate was loaded in each well.
GAPDH and α-Tubulin were used as controls for densitometry quantification. The quantified
data shown represent at least three independent experiments.

Cytofluorometric Analysis of Mitochondrial Membrane Potential

Mitochondrial membrane potential in diff-MN9D cells was measured as previously described
[30]. Briefly, MN9D cells were seeded into black-walled, clear-bottomed 24-well plates
onto Poly-L-Lysine (PLL) coated (Sigma, P2636, MW = 30,000-70,000, 1 mg/mL) Assistent
glass cover slips (12 mm, No.0, distributed by Carolina Biological Supply) at a density
of 35,000 cells per well in 500 μL CM. The MN9D cells were incubated overnight at
37°C, 5% CO2 and were then differentiated via a complete media change with DM. After 72 hrs in
DM, the diff-MN9D cell cultures were treated with C2-Cer or DMSO vehicle, or TNF or
media vehicle via a complete media change with 1X treatment in DM. After incubation
with C2-Cer for 18 hrs or TNF for 36 hrs, tetra methyl rhodamine methyl ester (TMRM)
(Invitrogen, T668, re-suspended in DMSO) was loaded into treated diff-MN9D cells at
150 nM in warm assay buffer (AB, 500 μL per well) which consisted of: NaCl (80 mM),
KCl (75 mM), D-glucose (25 mM) and HEPES (25 mM) diluted in sterile H2O and adjusted to a final pH of 7.4. To control for TMRM background cytofluorescence,
carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma, C2759) was used. At the time
of incubation with TMRM, 10 μM CCCP (re-suspended in DMSO) was co-added with TMRM
in AB to parallel wells of diff-MN9D cells treated with TNF or C2-Cer. TMRM and TMRM/CCCP
loaded cells were incubated for 15 minutes in a humidified incubator with 5% CO2 at 37°C prior to quantification of TMRM cytofluorescence by excitation at 544 nm
wavelength and emission at 590 nm wavelength on a FLUOstar Omega plate reader (BMG
Scientific). The TMRM signal in TMRM/CCCP conditions is considered background, and
this signal was used to normalize TMRM cytofluorescence values for each respective
TNF or C2-Cer condition.

Caspase Inhibition and BAPTA-AM Studies

Diff-MN9D cells in 96-well plates were treated in triplicate or quadruplicate with
TNF or C2-Cer alone or were co-treated with one of two caspase inhibitors, 25 μM Z-VAD-FMK
(Z-VAD, a pan caspase inhibitor, obtained from Promega), or 25 μM Z-IETD-FMK (Z-IETD,
a caspase 8-specific inhibitor, obtained from R&D Systems). The treated diff-MN9D
cells were incubated at 37°C, 5% CO2 with C2-Cer for 24 hrs or with TNF for 48 hrs prior to determination of overall cell
viability via the MTS assay as described above. For BAPTA-AM studies, diff-MN9D cells
were pre-loaded with the cell permeant intracellular Ca2+ chelator BAPTA-AM (BAPTA, 10 μM) 30 min prior to treatment with concentrations of
C2-Cer. At the endpoint of the study, cell viability was assayed by MTS reduction.

Lipidomics for Quantitative Analysis of Complex Sphingolipids and Sphingoid Base

Cell Treatments with TNF, Ceramide and Sphingoid Bases

After incubation in DM for 72 hours, diff-MN9D cells cultured in 96-well plates were
treated in triplicate or quadruplicate by a 50% media change with DM that contained
2X TNF (recombinant mouse, R&D MT-410), C2-Ceramide (C2-Cer, N-acetyl-D-Sphingosine,
Sigma A7191) or C2-dihydroceramide (C2-DH-Cer, Sigma, C7980) as a negative control
for C2-Ceramide because it lacks the 4–5 trans bond in the sphingosine moiety and cannot activate downstream ceramide signaling
[22,32]. The TNF was dissolved in sterile Phosphate Buffered Saline (PBS, Sigma) and C2-Cer
and C2-DH-Cer were dissolved in DMSO (Sigma) and aliquotted and stored under argon
gas. As a control in parallel treatments, a DMSO vehicle condition equivalent to the
amount of DMSO in the highest concentration of C2-Cer/C2-DH-Cer was used. TNF, C2-Cer
or C2-DH-Cer-treated diff-MN9D cells were incubated at 37°C, 5% CO2 for 72 or 48 hrs respectively, prior to being evaluated for overall viability using
the MTS assay (described below). TNF, C2-Cer or C2-DH-Cer treatments of diff-MN9D
cells in 24-well or 6-well plates were done in duplicate or triplicate by a complete
media change from DM to DM containing 1X TNF, C2-Cer or C2-DH-Cer. Etanercept, an
Fc-fusion protein consisting of TNFR2 and the Fc component of human immunoglobulin
IgG1, was used as a positive control because it binds TNF and blocks its bioactivity
[10,33].

Lipid-BSA stock solutions of the following sphingolipids from Avanti Polar Lipids
were prepared as per published protocols [34,35]. 1-deoxysphinganine C18H39NO (Catalog # 860493), 1-desoxymethylsphinganine C17H37NO
(Catalog # 860473); 1-deoxysphingosine C18H37NO (Catalog# 860470); 1-desoxymethylsphingosine
C17H35NO (Catalog # 860477); C16 ceramide C34H67NO3 (Catalog # 860516); Sphingosine
(d18:1) C18H37NO2 (Catalog # 860490) and Sphinganine (d18:0) C18H39NO2 (Catalog #
860498). Briefly, lipids were placed in Pyrex 13x100 mm borosilicate, screw-capped
glass test tubes with Teflon caps and solubilized in a volume of ethanol to get a
final concentration of 100 mM; sonication and warm tap water were employed to ensure
homogenous resuspension. To make 1:1 (concentration) sphingoid base-BSA complex, 20
uL of a given sphingoid base (100 mM) in ethanol was quickly injected into a 1 mL
volume of a BSA (2 mM) solution by using a Hamilton syringe. To ensure optimal complexing
of the lipids to BSA, tubes were shaken vigorously and sonicated as needed. When treating
the diff-MN9D cells, different concentrations of sphingoid base-BSA complexs were
prepared in differentiation media and added to diff-MN9D cells and incubated for 24 hours
at the concentrations indicated under “Results”. When treating the rat MES cultures,
sphingoid base-BSA complexes were prepared in treatment media (DMEM/Ham F-12 with
1% Pen/strep, 1% Glutamine, 1% non-essential amino acids and 2.5% FBS) without bFGF.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism5 software (GraphPad Prism,
San Diego, CA). Intergroup differences among the means between the various dependent
variables were analyzed using one-way ANOVA; when ANOVA indicated significant differences,
it was followed by Tukey’s post-hoc group comparison test. Differences among group means between two independent variables
were analyzed by two-way ANOVA, followed by Tukey’s post-hoc test when the ANOVA indicated significant differences. Values expressed are the group
means +/- standard error of the mean (SEM).

Results

In light of our previous findings showing that ventral mesencephalon dopaminergic
(DA) neurons are acutely sensitive to TNF in vitro and in vivo[10], we hypothesized that ceramide sphingolipids are critical effectors of TNF-induced
cytotoxicity. First, we aimed to establish a correlation between TNF-dependent ceramide
generation and the effect of TNF or ceramide exposure on the viability of neuronally
differentiated MN9D cells or primary DA neurons. We found that TNF dose-dependently
decreased the viability of diff-MN9D cells as measured by the MTS metabolic assay
(Figure 1A). To test the hypothesis that elevated ceramide is directly toxic to diff-MN9D cells,
we treated the cells with various concentrations of C2-Cer or C2-dihydroceramide (C2-DH-Cer)
as a negative control; C2-DH-Cer is an analog of C2-Cer lacking the 4–5 trans bond in the sphingosine moiety that is incapable of activating downstream ceramide
signaling [22,32]. We found that C2-Cer but not C2-DH-Cer induced dose-dependent decreases in diff-MN9D
viability (Figure 1B). We previously determined that non-differentiated MN9D (non-diffMN9D) cells are
not sensitive to concentrations of TNF that elicit cytotoxicity in diff-MN9D cells
[26]. Similarly, C2-Cer was not cytotoxic to non-diff-MN9D cells (Additional file 1: Figure S1).

TNF-induced neurotoxicity in DA cells and neurons is attenuated by SMase inhibitors

Ceramide can be generated either through a de novo biosynthesis pathway involving several enzymatic reactions downstream of the initial
condensation of serine and palmitoyl-CoA on the cytoplasmic surface of the ER or through
the sphingomyelin recycling pathway whereby acid or neutral sphingomyelinases (SMases)
hydrolyze sphingomyelin (SM) to ceramide [36]. We hypothesized that activation of SMases at the plasma membrane by the activated
TNFR1/TNF receptor complex is the mechanism by which TNF exposure leads to ceramide
signaling and cytotoxicity in DA cells. To test this hypothesis directly, we pre-treated
diff-MN9D cells with different inhibitors of SMases for 30 minutes followed by treatment
with TNF for 48 hrs. We pre-treated diff-MN9D cells with three different compounds
that inhibit SMases with different mechanisms of action. Pre-incubation with desipramine
(Des) (an inhibitor of acid sphingomyelinase, ASMase[37]), GW4869 (an inhibitor of neutral sphingomylinase, NSMase [38]), or with 7c, also known as ARC39 (an inhibitor of lysosomal and secreted ASMase
[39]) at the concentrations indicated all significantly attenuated TNF-induced cytotoxicity
of diff-MN9D cells as measured by the MTS assay (Figure 2A). To confirm and extend these findings, we assayed the extent to which two of these
SMase inhibitors attenuated TNF-induced death of DA neurons in primary neuron-glia
cultures from rat ventral mesencephalon. Consistent with the results in MN9D cells,
Des and GW4869 protected primary DA neurons from TNF-induced death (Figure 2B) to an extent comparable to that achieved in previous studies using the soluble
TNF-selective inhibitor XENP345 [10]. Together these pharmacological data strongly suggest that TNF-dependent activation
of SMases results in SM hydrolysis and generation of ceramide that is cytotoxic to
DA neurons, compromising their viability. To confirm that the ceramide-generating
pathway involved in mediating TNF-dependent cytotoxicity is due to SM hydrolysis by
SMases rather than through de novo ceramide formation, we repeated these experiments using pharmacological inhibitors
of the de novo ceramide biosynthesis pathway. We observed that inhibition of the enzyme serine palmitoyltransferase
(the rate-limiting enzyme in de novo ceramide biosynthesis) by myriocin or inhibition of the enzyme ceramide synthase
(which converts sphinganine to dihydroceramide) by Fumonisin B1 did not mitigate TNF-induced
cytotoxicity in diff-MN9D cells (Additional file 2: Figure S2). Collectively, our data support a model in which SMase hydrolysis of
SM to form ceramide is requisite for TNF-induced cytotoxicity in diff-MN9D cells and
DA neurons.

Additional file 2.Figure S2. TNF-induced cytotoxicity in diff-MN9D cells does not require de novo biosynthesis
of ceramide. Diff-MN9D cells were pre-treated with myriocin (Myr, an inhibitor for
serine palmitoyltransferase) or fumonisin B1 (FB1, an inhibitor of ceramide synthase)
for 30 minutes prior to addition of TNF for an additional 48 hours. Cell viability
was measured via an MTS assay as described under Methods. All values represent group
means +/− SEM, n =3 - 4. Two-way ANOVA to test for differences between TNF with inhibitor
versus TNF without inhibitor for both Myrocin and Fumonisin B1; there were no significant
differences between No MYR and 10 μM MYR at any TNF concentration, as determined by
a two-way ANOVA. There was statistically significant TNF-induced death of diff-MN9D
cells, as determined by a Tukey’s post-hoc test following a statistically significant
one-way ANOVA, where ** denotes p < 0.01, *** p < 0.001 for No MYR conditions relative
to vehicle, * denotes p < 0.05 for TNF 10 + No MYR compared to TNF 30 + No MYR, and
### denotes p < 0.001 for 10 μM MYR conditions relative to vehicle. There were no
significant differences between No FB1 and 50 μM FB1 at any TNF concentration, as
determined by a two-way ANOVA. There was statistically significant TNF-induced death
of diff-MN9D cells, as determined by a Tukey's post-hoc test following a statistically
significant one-way ANOVA where * denotes p < 0.05, *** denotes p < 0.001 for No FB1
conditions relative to vehicle, ** denotes p < 0.01 for TNF 10 + No FB1 compared to
TNF 30 + No FB1, and ### denotes p < 0.001 for 50uM FB1 conditions relative to vehicle,
# denotes p < 0.01 for TNF 10 + 50 μM FB1 compared to TNF 30 + 50 μM FB1.

TNF and ceramide have been shown to impinge on ER stress mechanisms in non-neuronal
cells types [40,41] and ER stress has been implicated as a potentially important pathway in PD pathogenesis
[42], being coupled to the cell death program in DA cells in response to the toxin paraquat
[43]. Therefore, we investigated the extent to which activation of ER stress pathways
by TNF are dependent on ceramide generation by SMase activity in diff-MN9D cells.
We used immunoblots to ascertain if TNF treatment of diff-MN9D cells increased protein
expression of key ER stress transducers, including activating transcription factor
6 (ATF6), ER- resident PKR-like eIF2α kinase (PERK), and inositol requiring enzyme-1
(IRE). We found that the increased expression of ER stress proteins by TNF and C2-Cer
was comparable to increased protein levels caused by the positive control tunicamycin,
(Figure 3) which is known to potently induce ER stress by inhibiting protein N-glycosylation
[44]. These results support a model in which TNF employs ceramide signaling to elicit
ER stress in DA cells.

TNF has been reported to cause rapid decreases in mitochondrial membrane potential
and coincident increases in reactive oxygen species [45]. Consistent with our hypothesis that ceramide is an important downstream effector
of TNF cytotoxicity, ceramide itself has been shown to directly affect the mitochondrial
electron transport chain [46]. To further elucidate the mechanisms of TNF and C2-Cer-induced cytotoxicity and to
determine if TNF/ceramide signaling in diff-MN9D cells impinges on mitochondria, we
investigated whether TNF or C2-Cer adversely impact mitochondrial membrane potential
by evaluating tetramethyl rhodamine methyl ester (TMRM) cytofluorescence. TMRM is
a cationic mitochondrial-selective probe that accumulates in the negatively charged
mitochondrial membrane in proportion to mitochondrial membrane potential. Diff-MN9D
cells treated with 5 ng/mL TNF for 36 hrs or 5 or 10 μM C2-Cer for 18 hrs exhibited
compromised mitochondrial membrane potential as evidence by reduced TMRM cytofluorescence
relative to vehicle treated diff-MN9D cells (Figure 4A), lending support to the interpretation that both TNF and C2-Cer adversely affect
mitochondrial integrity in diff-MN9D cells. Moreover, the SMase inhibitors desipramine
and GW4869 partially restored the TMRM signal in diff-MN9D cells (Figure 4A). To confirm and extend these findings we performed an additional assay to measure
TNF-induced cytotoxicity. Diff-MN9D cells were treated for 18 hrs with 5 or 10 μM
C2-Cer or 5 ng/mL TNF; lactate dehydrogenate (LDH) release was then measured. In agreement
with results from MTS assays, pre-treatment with SMase inhibitors (Desipramine or
GW4869) attenuated TNF-induced LDH release. The TNF inhibitor etanercept was used
as a positive control. These data support a model in which TNF-induced cytotoxicity
is mediated via ceramide-dependent signaling leading to disruption of mitochondrial
membrane potential in DA cells.

Loss of mitochondrial membrane potential and release of cytochrome C from mitochondria
generally precede caspase-dependent apoptotic cell death and a wealth of data has
linked TNF bioactivity to caspase activation and apoptosis in various cell types (reviewed
in [47]). Similarly, ceramide has been reported to cause apoptotic cell death by altering
the Bax/Bcl2 ratio which triggers cytochrome C release from the mitochondria and results
in activation of the caspase-9/-3 cascade in C6 glioma cells [48]. Therefore, we investigated the extent to which addition of SMase inhibitors during
TNF treatment attenuated caspase signaling. Western blot analyses showed that desipramine
and GW4869 significantly attenuated caspase 3 cleavage in TNF-treated diff-MN9D cells
(Figure 5A, B). To correlate this finding with TNF-induced cytotoxicity in diff-MN9D cells,
we determined the extent to which pan-caspase inhibition (with Z-VAD) or caspase 8
inhibition (with Z-IETD) could ameliorate TNF dose-dependent loss of viability in
diff-MN9D. We found that both caspase inhibitors robustly protected diff-MN9D cells
from TNF-induced cytotoxicity at all TNF concentrations (Figure 5C), demonstrating that caspase activation is obligate for TNF-induced apoptotic cell
death in terminally differentiated MN9D cells and suggesting that TNF-dependent ceramide
generation promotes activation of caspase 8 and caspase 3 signaling cascades that
lead to apoptotic death in DA cells and neurons. Interestingly, we also found that
C2-Cer-induced cytotoxic cell death in diff-MN9D cells was not significantly blocked
by Z-VAD or Z-IETD (Figure 6A), which is not entirely surprising since exogenously added C2-Cer would act downstream
of TNF/TNFR1-dependent caspase 8 activation. However, we hypothesized that TNF-stimulated
ceramide exerts cytotoxicity in DA cells by dysregulating intracellular Ca2+ based on reports that implicate defective Ca2+ homeostasis in apoptotic cell death of neuronal populations induced by aberrant sphingolipid
metabolism [49]. To test this hypothesis directly, we pre-incubated diffMN9D cells with BAPTA-AM
prior to exposure to C2-Cer and found that buffering intracellular free calcium nearly
ablates C2-Cer-induced toxicity in diff-MN9D cells (Figure 6B), suggesting that elevation of [Ca2+i contributes to C2-Cer-induced neurotoxicity.

Next, we tested the hypothesis that TNF-dependent ceramide-induced cytotoxicity in
diff-MN9D cells may also result from reduced activation of pro-survival pathways,
such as Akt signaling. Therefore, we investigated the effect of TNF on phosphorylation
of Akt, a key step in pro-survival signaling in the majority of neurons [50,51] We found that TNF treatment reduced p-Akt levels in DA cells and SMase inhibitors
robustly blocked this effect (Figure 7). Together with results from caspase inhibition experiments, these data suggest that
TNF treatment leads to generation and accumulation of ceramide (and perhaps other
downstream sphingolipid metabolites), leading to cytotoxicity in DA neurons via increased
ER stress, compromised mitochondrial membrane potential, increased caspase-3 dependent
apoptotic signaling cascades, and attenuation of phospho-Akt-dependent pro-survival
signaling.

Given that SMase inhibition affords significant protection from TNF-dependent toxicity
in DA neuroblastoma cells and primary DA neurons, it was of interest to confirm that
TNF treatment results in detectable formation of ceramide in vivo. We used a lipidomics approach to enable quantitative analysis of complex sphingolipids
and sphingoid bases in lipid extracts of MN9D cells exposed to PBS or soluble TNF
for up to 48 hours. We chose to use DA neuroblastoma cells for our analysis because
a homogeneous population of cells is needed for a meaningful result and primary DA
neurons only make up a small percentage of total neurons in ventral midbrain cultures.
Our analyses indicated that TNF exposure significantly increased the intracellular
levels of total ceramide (Cer), sphingomyelin (SM), and hexosylceramide (HexCer) (Figure
8A) as well as several sphingoid bases including sphingosine (So), sphinganine (Sa),
sphingosine-1-P (SoP), sphinganine-1-P (SaP), and the atypical sphingoid bases deoxy-sphinganine
(deoxySa or DEOSA) and desoxymethylsphinganine (desoxyMeSa or DEOMSA) (Figure 8B). TNF-induced increases in the levels of other complex sphingolipids including deoxydihydro-Ceramide
(deoxyDH-Cer) and deoxyceramide (deoxyCer) were not consistently or reproducibly detected
(data not shown). These data raise the possibility that in addition to ceramide, any
of these additional sphingolipids could be critical second messengers involved in
mediating TNF cytotoxicity in DA neuroblastoma cells.

Based on results from lipidomics analyses (Figure 8B) which indicated that TNF exposure not only increased ceramide levels but also resulted
in significant increases in the intracellular levels of several atypical deoxy-sphingoid
bases (DSBs), including deoxysphinganine (deoxySa or DEOSA) and desoxymethylsphinganine
(desoxyMeSa or DEOMSA), we wanted to test these atypical DSBs for direct cytotoxic
effects on cells. These DSBs are devoid of the C1-hydroxyl group of sphinganine and
can therefore neither be metabolized to complex sphingolipids nor degraded by the
regular sphingolipid catabolism, raising the possibility that they may accumulate
within DA neurons and may be cytotoxic. Therefore, we tested the extent to which 1-deoxySa,
1-desoxyMeSa, and 1-desoxyMeSo induce dose-dependent cytotoxicity in diff-MN9D cells
and found that all three induced dose-dependent cytotoxicity with an IC50 around 15
μM (Figure 9). To confirm and extend the significance of these findings, we investigated the cytotoxicity
of these atypical sphingoid bases on primary cultures from rat ventral mesencephalon.
We found that only 1-deoxySa significantly reduced the number of neuritic branches
and outgrowths per DA neuron at concentrations as low as 0.5 μM (Figure 10); a trend towards compromising DA neuron viability was also evident but it did not
reach statistical significance. No significant cytotoxic effects on primary DA neurons
by 1-desoxyMeSa and 1-desoxyMeSo were observed (Additional file 3: Figure S3).

Additional file 3.Figure S3. The atypical sphingoid bases 1-deoxyMeSa and 1-deoxyMeSo did not exert cytotoxicity
on primary DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon
were plated in 96-well plates and exposed to treatment media alone without BSA (0)
or to 1-desoxymethylsphingosine (1-desoxyMeSo) or 1-desoxymethylsphinganine (1-desoxyMeSa)
at the concentrations indicated in a complex with BSA (25 μM) for 48 hours prior to
assessing number of branches per cell, number of processes, and number of outgrowths
per cell as well as cell number using Image Xpress high-content imaging analyses.
All values represent group means +/− SEM, n = 3–4. There were no significant effects
from treatment with 1-deoxyMeSa and 1-deoxyMeSo as determined by a one-way ANOVA.

Figure 10.The atypical sphingoid base 1-deoxySa reduced neuritic branches and outgrowth in primary
DA neurons. Primary neuron-glia cultures from rat ventral mesencephalon were plated in 96-well
plates and exposed to treatment media alone without BSA (0) or to 1-deoxysphinganine
(1-deoxySa) at the concentrations indicated in a complex with BSA (25 μM) for 48 hours
prior to assessing number of branches per cell, number of processes, and number of
outgrowths per cell as well as cell number using Image Xpress high-content imaging
analyses. 1-DeoxySa was the only one of the sphingoid bases tested that reduced neurite
outgrowth and branching. All values represent group means +/− SEM, n = 3–4. One-way
ANOVA with Tukey’s post-hoc’ * denotes difference from treatment media alone at p < 0.05,
and ** denotes p < 0.01. N.S. denotes not significant.

Discussion

The purpose of these studies was to test the hypothesis that ceramide-dependent signaling
mediates TNF-induced cytotoxicity and degeneration of DA neurons. Our results indicate
that exposure of neurally differentiated DA neuroblastoma cells to soluble TNF induced
activation of membrane-bound sphingomyelinases (SMases) and sphingomyelin (SM) turnover
resulting in generation of ceramide as measured by lipidomics mass spectrometry. Direct
addition of C2-ceramide to DA neuroblastoma cells or primary DA neurons in vitro resulted in dose-dependent cytotoxicity, and pharmacological inhibition of SMases
with three different inhibitors of SMase function to block ceramide generation during
TNF exposure (but not inhibitors of de novo ceramide synthesis) afforded significant protection from TNF-induced cytotoxicity.
Although desipramine can exert SMase-independent effects on cells [52], two other inhibitors with greater specificity for SMase (GW4869 and ARC39) afforded
similar protection against TNF-induced cytotoxicity. Based on these findings, we propose
a model by which binding of soluble TNF to TNFR1 on the cell surface of DA neurons
activates SMases to generate ceramide and trigger downstream signaling cascades that
compromise survival of DA neurons by eliciting ER stress, reducing mitochondria membrane
potential, leading to activation of caspase-3-dependent pro-apoptotic signaling and
inhibition of Akt-dependent pro-survival signaling cascades which combine to compromise
survival of DA neurons (Figure 11). Interestingly, TNF treatment also induced SM biosynthesis (Figure 8A); the significance of this novel finding is unknown, but TNF and lipopolysaccharide
(LPS) have both been reported to induce sphingolipid biosynthesis in liver [53] and macrophages [54]. It is also worth noting that increases in atypical deoxy-sphingoid bases (DSBs)
were detectable in DA cells after prolonged exposure to TNF (Figure 8B), the potential significance of which is discussed below.

Figure 11.Proposed model for cellular mechanisms and signaling pathways activated by TNF and
ceramide/sphingolipid signaling to induce neurotoxicity in DA neurons. We propose a model by which TNF/TNFR1-dependent activation of SMases triggers production
of ceramide and other downstream lipid metabolites that promote activation of caspase-8/3
signaling, decreased Akt activation and mitochondrial membrane potential, and increased
endoplasmic reticulum (ER) stress in DA cells.

Glycosphingolipid (GSL) metabolism represents a metabolic cross point that interconnects
lipid (acyl-CoA) and amino acid (serine and alanine) metabolism. For a detailed review
of the metabolic interrelationships that account for the tens of thousands of molecular
subspecies in the mammalian sphingolipidome, the reader is referred elsewhere [55,56]. Briefly, ceramide (Cer) consists of a fatty acid acyl chain that varies in length
and saturation, and a sphingoid base that differs in the number and position of double
bonds and hydroxyl groups. Tissue- and cell type-specific ceramide synthases control
the length of the fatty acid chain of ceramide. Sphingoid bases are formed from the
precursors L-serine and palmitoyl-CoA in a reaction catalyzed by serine-palmitoyltransferase
(SPT). SPT metabolizes other acyl-CoAs besides palmitoyl-CoA but also shows variability
towards the use of other amino acid substrates. For instance, SPT is also able to
metabolize alanine, which results in the formation of an atypical deoxy-sphingoid
base (DSB). These atypical and relatively novel DSBs are devoid of the C1-hydroxyl
group of sphingosine (SA) and are therefore neither metabolized to complex sphingolipids
nor degraded by the regular sphingolipid catabolism, since sphingosine-1P as a catabolic
intermediate cannot be formed from DSBs [34,57]. Missense mutations in SPT long-chain subunit 1 (SPTLC1) increase its promiscuous activity towards alanine over serine and result in pathologically
elevated DSB levels in the case of the autosomal dominant hereditary sensory and autonomic
sensory neuropathy type 1 HSAN1 [34,58]; as evidence of their capacity to induce cytotoxicity, addition of deoxySa to dorsal
root ganglion (DRG) neurons in culture can be shown to reduce neurite formation and
to disrupt the neuronal cytoskeleton [34]. Given that we observed similar effects in deoxySa-treated DA neurons, we speculate
that TNF-stimulated de novo synthesis of atypical DSBs may be a secondary mechanism that contributes to TNF-dependent
toxicity and reduced viability of DA neurons during inflammatory stress. In fact,
neurons may have heightened vulnerability to cellular disturbances in lipid metabolism
based on the observation that the majority of GSL lysosomal storage diseases (LSDs)
with CNS involvement result in neuronal death, even though the enzymes affected by
the gene mutations are expressed ubiquitously [59].

TNF and ceramide have been shown to impinge on endoplasmic reticulum (ER) stress mechanisms
in non-neuronal cells types [40,41] and ER stress has been implicated as a potentially important pathway in neurodegenerative
diseases [60]; however, whether ER stress is a cause, result, or epiphenomenon in the DA neuron
loss that occurs in PD has not been firmly established. ER stress-mediated cell death
has been implicated in PD pathogenesis [42,61], being coupled to the cell death program in DA cells in response to the toxin paraquat
[43]. Here, we show for the first time that inflammatory signaling through TNF and ceramide
induces ER stress in DA neuron-like cells and that SMase inhibition attenuates ER
stress and prevents TNF-induced cytotoxicity (as measured independently by MTS and
LDH release assays). ATF6 is a direct target of the ER stress response [61] and is known to activate transcription of chaperone proteins [62] to facilitate protein folding and processing capacity; ATF6 also activates ER-associated
degradation (ERAD) to promote the degradation of terminally misfolded proteins [63]. Mechanistically, defective calcium homeostasis, especially increased intracellular
Ca2+ release, presumably from the ER, has been implicated in neuronal cell death in mouse
models exhibiting increased CNS glucosylsphingosine levels which can also suppress
neuronal outgrowth [49,64,65]. Our data that BAPTA-AM markedly blocked ceramide-induced neurotoxicity is consistent
with a role for ceramide as a disruptor of Ca2+ homeostasis in DA neurons. Interestingly, a recent study reported that MPTP treatment
induced ER stress and decreased AKT phosphorylation via loss of TRPC1-dependent ER
Ca2+ homeostasis in human dopaminergic neuroblastoma SH-SY5Y cells [66]. Importantly, signs of TNF pathway activation [67,68], ER stress [69,70] and reduced levels of AKT phosphorylation [71] have all been reported in the SNpc of PD patients. Taken together, these findings
support the idea that disrupted ER Ca2+ homeostasis and compromised Akt pathway activation is a common mechanism by which
TNF-dependent inflammation and oxidative neurotoxins compromise survival of DA neurons
and lead to development of PD-like features.

Many of the genes associated with PD implicate aberrant mitochondrial function in
disease pathogenesis [72] and MPTP and rotenone, which are commonly used in rodents to induce features of parkinsonism,
are potent mitochondrial complex I inhibitors [73]. While compromised mitochondrial function has been strongly implicated in PD pathophysiology
[72], to date, compromised mitochondrial membrane potential in response to inflammatory
stimuli (in this case TNF and C2-Cer) has never been demonstrated in DA cells or DA
neurons. Our data demonstrate that TNF and C2-Cer-induced cytotoxicity in diff-MN9D
cells correlates closely with reduced mitochondrial membrane potential and treatment
with SMase inhibitors reverses these mitochondrial deficits. Similarly, in NGF-differentiated
PC12 cells, ceramide signaling has been reported to increase mitochondrial Ca2+ levels and to induce ultrastructural alterations [74]. Furthermore, ceramide-induced increases in mitochondrial free calcium were subsequently
shown to originate in the ER in a ROS-independent fashion [75]. Our data showing that BAPTA-AM buffering of intracellular free calcium ablates ceramide-induced
cytotoxicity in diff-MN9D cells support this kind of model; however, additional studies
are needed to determine the source of the cellular Ca2+ and/or the extent to which disrupted ER or mitochondrial Ca2+ homeostasis plays a causative or synergistic role in TNF or GSL-induced mitochondrial
dysfunction in DA neurons [49,64,65].

A role for caspase-dependent apoptotic signaling has been implicated in the death
of DA neurons that occurs in PD [76,77] and our findings strongly support a role for caspase 8/caspase 3 signaling as downstream
effectors in TNF-dependent death of dopaminergic cells. It should be noted that we
observe distinct differences in the overall requirement for caspase signaling in TNF-
versus C2-Cer-dependent cytotoxicity in diff-MN9D cells. One reason for this may be
that TNF signaling generates ceramide in a physiological range which acts in concert
with other TNF receptor-mediated signaling events to trigger downstream caspase-dependent
apoptotic processes, whereas addition of exogenous C2-Cer (at concentrations that
may not be within a physiological range) artificially bypasses TNF receptor-mediated
events and exerts toxic effects by targeting other pathways in addition to mitochondria
and caspase inhibition and is not sufficient to attenuate cytotoxicity from this extreme
insult. Jurkat T cells require ASMase translocation to plasma membrane lipid microdomains
to elicit localized ceramide production and eventual apoptotic cell death [78]. Interestingly, in these cells, ASMase translocation has been shown to occur via
two distinct mechanisms: a caspase-dependent mechanism utilized by Fas-L and a previously
unrecognized caspase-independent mechanism elicited by short wave ultraviolet irradiation
(UV-C). Specifically, it was determined that the caspase-independent mechanism of
ASMase translocation led to cell death of Jurkat cells and that UV-C treatment of
Jurkat cells activates the sphingomyelin pathway independent of caspase 8 or in the
presence of a pan-caspase inhibitor. In this study, the authors note that while ASMase
is not a direct target of caspase 8, surface translocation of ASMase activated by
Fas-L or other TNF superfamily ligands requires minimal caspase 8 and FADD activation
(~2% activation is sufficient). In the case of diff-MN9D cells, exogenous addition
of C2-Ceramide bypasses the step of ASMase translocation to lipid microdomains in
the plasma membrane as well as the concomitant activation of caspase cascades from
the signaling complex assembled in microdomains at the cell membrane that otherwise
occurs in response to TNFR1 activation, which is likely to result in toxicity that
is caspase-independent. Alternatively, it is possible that exogenous addition of ceramide
is sufficient to elicit caspase independent cell death via release of mitochondrial
apoptogenic factors, but that engagement of TNFR1 by its ligand TNF leads to SMase-dependent
production of ceramide and caspase-dependent cell death of diff-MN9D cells. Lastly,
Deerberg and colleagues report that there is a combined requirement of both the ER
and mitochondria in the induction of signaling pathways of ceramide-mediated caspase-independent
programmed cell death in Jurkat cells [79] and a similar mechanism may be occurring in C2-Ceramide treated diff-MN9D cells.
Collectively, our data support a model whereby TNF concentrations in the range that
elicit half-maximal cytotoxicity and that correspond to low TNF receptor 1 (TNFR1)
occupancy activate SMase to initiate downstream signaling by ceramide and other sphingolipid
metabolites, which trigger ER stress, decreased mitochondrial membrane potential,
and eventually culminate in the caspase-dependent cytotoxic cell death of DA neurons
(Figure 11). Support for this model comes from the multiple studies presented here in which
pharmacological inhibition of SMases to block ceramide generation during TNF exposure
maintained mitochondrial membrane potential, markedly attenuated TNF-induced ER stress
and caspase signaling and restored p-Akt levels in DA cells, thereby promoting significant
protection from TNF-induced neurotoxicity.

The histopathophysiological hallmark of Parkinson’s disease (PD) is the formation
of intraneuronal aggregation and clustering of α-synuclein and ubiquitinated proteins
into inclusions commonly referred to as Lewy bodies (LB) typically found in DA neurons
of the substantia nigra pars compacta (SNpc) in the ventral midbrain [80]. Notably, several genes known to be involved in the genetics of Lewy body disease
(LBD) or heritable PD share in common the fact that they impinge on ceramide metabolism
[81]. Therefore, ceramide metabolism has recently received attention as an emerging pathway
involved in LBD [82]. For example, heterozygous loss-of-function mutations of the glucocerebrosidase (GBA)
locus have recently been shown to be a potent risk factor for PD [81,83]. GBA catalyzes the dissolution of glucocerebrosidase to ceramide and glucose. The
lysosomal storage disease Gaucher’s disease (GD) arises from homozygous mutations
in GBA, leading to extreme lysosomal accumulation of GBA substrates and onset of GD
symptoms [84]. Interestingly however, GBA substrates do not significantly accumulate in the lysosomes
of patients with heterozygous GBA mutations, lending support to the hypothesis that
generally disrupted ceramide metabolism, as opposed to specific loss of GBA function,
may be an initiating factor in PD [81]. Our data offer a mechanistic link between specific GSL accumulation, ER stress,
mitochondrial dysfunction, apoptotic signaling and neuronal death in dopaminergic
neurons in response to TNF exposure which may be of significance in PD but perhaps
also in other chronic neurodegenerative conditions characterized by elevated levels
of TNF and other inflammatory factors. Interestingly, the ASMase inhibitor desipramine
induces specific and rapid intracellular degradation of ASMase and concomitant abolishment
of enzymatic activity [85]; however, desipramine is a tricyclic antidepressant and its action on neurotransmitters
seems to be independent of its effects on ASMase activity. Nevertheless, desipramine
has been used in clinical trials to treat depression in PD patients [86]; these trials were very short-lived however, and the effect of desipramine on ceramide
signaling was not evaluated as an outcome. Therefore, our data and the data of other
groups associating ceramide biology and metabolism with PD warrant future studies
examining the potential neuroprotective effects of inhibition of ASMase or NSMase
in animal models of PD.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

TNM designed and performed experiments with MN9D cells to measure cell viability,
ER stress, mitochondria membrane potential, and caspase activation; analyzed data
and performed statistical analysis; participated in writing and editing of manuscript.
XC designed and performed experiments with MN9D cells and primary DA neuron cultures
to measure cell viability, ER stress, mitochondria membrane potential, caspase and
Akt activation, sphingolipid measurements, and atypical sphingoid base toxicity studies;
analyzed data and performed statistical analysis; participated in writing and editing
of manuscript. SP performed lipidomics experiments and data analysis; participated
in writing and editing of manuscript. AHM participated in preparation of lipid-BSA
complexes, directed lipidomics experiments and interpretation of data; participated
in writing and editing of manuscript. MGT participated in study design, interpretation
of data, writing and editing the manuscript. All authors read, edited, and approved
the final manuscript.

Acknowledgements

We would like to thank Dr. William Holland at UT Southwestern for helpful discussions
regarding ceramide biology and members of the Tansey lab for helpful discussions.
We would also like to thank Dr. Philip Scherer at UT Southwestern for kindly providing
myriocin for our studies and Dr. Christoph Arenz at Humboldt University in Berlin
for providing us ARC39 for our studies. This work was made possible by generous funding
from the National Institutes of Health NINDS 5R01NS049433 (MGT), a National Institutes
of Health Pre-doctoral NRSA Training Grant GM007062 (TNM), and GM069338 to support
the Sphingolipids Core of Lipid Maps (AHM).

Part of this work was completed by Terina N. Martinez to fulfill the requirements
for a doctoral thesis.